Calmodulin binds the N-terminus of the functional amyloid Orb2A inhibiting fibril formation

The cytoplasmic polyadenylation element-binding protein Orb2 is a key regulator of long-term memory (LTM) in Drosophila. The N-terminus of the Orb2 isoform A is required for LTM and forms cross-β fibrils on its own. However, this N-terminus is not part of the core found in ex vivo fibrils. We previously showed that besides forming cross-β fibrils, the N-terminus of Orb2A binds anionic lipid membranes as an amphipathic helix. Here, we show that the Orb2A N-terminus can similarly interact with calcium activated calmodulin (CaM) and that this interaction prevents fibril formation. Because CaM is a known regulator of LTM, this interaction could potentially explain the regulatory role of Orb2A in LTM.


Introduction
Cytoplasmic polyadenylation element-binding (CPEB) proteins are important mRNA translational regulators. In Drosophila melanogaster, the CPEB homolog Orb2 has been shown to be important for long-term memory (LTM) by regulating mRNA translation at the synapse [1][2][3]. There are two isoforms of Orb2, Orb2A and Orb2B. Both are present in the synapse, though Orb2B is found abundantly throughout the cytoplasm whereas Orb2A concentrations are low [3]. Although both Orb2A and Orb2B can form cross-β (amyloid) fibrils in vitro, Orb2A is required for Orb2 aggregation in vivo and both isoforms are found together in in vivo aggregates [3][4][5][6]. This aggregation is necessary for long-term memory [3]. Much of the work on Orb2 aggregation in memory suggests that Orb2A may be a key regulator of overall Orb2 aggregation [2,3,7]. The formation of functional fibrils is thought to be a highly regulated process, in contrast to cross-β fibrils in disease. However, the mechanism that regulates Orb2A aggregation is currently not known.
Orb2A and Orb2B differ only in their N-termini. Orb2A has only 8 N-terminal residues before the first residue shared with Orb2B. The N-terminus of Orb2B is rich in serine and has over 150 residues before the first common residue with Orb2A ( Fig 1A). Both Orb2A and Orb2B share a glutamine/histidine-rich (Q/H-rich) domain followed by a glycine-rich domain, two RNA binding domains, and a C-terminal zinc finger. The cryo-EM structure of functional Orb2 fibrils extracted from Drosophila brains by Hervás and co-workers showed that the Q/H-rich domain of Orb2 forms the cross-β core [5]. However, the N-terminus of a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Orb2A, which is not part of this core, was shown to be important for Orb2 fibril formation and LTM: Using random mutagenesis, Majumdar and co-workers showed that the first eight amino acids of Orb2A were over-represented as mutations that inhibited aggregation [3]. Point mutations of residue F5 in Orb2A affected aggregation in vitro and LTM in flies [3,4]. Using solid-state NMR and electron paramagnetic resonance (EPR), we showed that fibrils formed by the first 88 amino acids of Orb2A had their cross-β core located within these first residues. In fact, the first 21 residues of Orb2A, which precede the Q/H-rich domain, were able to form cross-β fibrils on their own [8]. In addition to being able to form cross-β fibrils, the N-terminal 16 residues of Orb2A form an amphipathic helix when plotted on a helical wheel (see Fig 1A). The combination of an amphipathic helix with a glutamine-rich domain makes Orb2A reminiscent of huntingtin exon-1 (HTTex1) in which an amphipathic N-terminus (N17) precedes a polyQ domain. Based on the fact that the HTTex1 N17 domain was shown to bind lipid membranes [9,10], we originally investigated the lipid binding capabilities of Orb2A. We showed that the N-terminus of Orb2A forms an amphipathic helix in the presence of anionic lipid vesicles and binds to these vesicles. This binding in turn inhibited the formation of the N-terminal cross-β fibril core [11].
A protein that commonly interacts with amphipathic helices is the calcium-sensing protein calmodulin (CaM). Among its many functions in a large number of different cell types, CaM is an important signal integration protein in neurons for LTM [12]. CaM is present in very high concentrations in the central nervous system, up to 10-100 μM [13], and has been shown to directly regulate several proteins involved in long-term potentiation (LTP). These include adenylyl cyclases AC1 and AC8 [14,15], calcineurin, and CaM kinases (CaMK) II and IV [16][17][18][19][20]. Depending on the level of Ca 2+ present, CaM can activate proteins specifically for LTP or long-term depression (LTD) [21], both of which are important in LTM. CaM interacts with sequences that are both amphipathic and positively charged. While there are many modes of binding with CaM depending on the target (reviewed in [22]) the most frequently observed A) Domain layout of Orb2A and Orb2B, including the amphipathic helix at the N-terminus of Orb2A. The extended N-terminus of Orb2B is highlighted in gray, the amphipathic region in purple, the Q/H-rich domain in orange, the G/S-rich domain in green, and the RBD in pink. B) Sequence of the N-terminal 88 amino acids of Orb2A used throughout this study. The colors correspond to the domains in A. C) Mechanism by which Ca 2+ /CaM binds to amphipathic helices. Blue circles represent Ca 2+ , the red helix is the linker domain of CaM, the purple helix is a hypothetical CaM binding partner. D) CaM binding site prediction using the calmodulin meta-analysis predictor. Overall per residue score that is equal to the number of times a residue is found in a canonical CaM-binding motif is shown in the first line followed by the Orb2A 1-88 sequence with the identified potential binding motif highlighted in yellow. Matches to canonical CaM binding motifs are shown at the bottom.
https://doi.org/10.1371/journal.pone.0259872.g001 mode relies on the binding of Ca 2+ to each of the four EF hand domains present in CaM. These four EF hands are separated, two to each side, by an unstructured linker, which becomes α-helical when Ca 2+ binds to the EF hands ( Fig 1C). This creates hydrophobic binding pockets with which many CaM binding partners interact.
In hippocampal dendrites, the CaM/Ca 2+ -regulated kinase CaMKII is responsible for activating mouse CPEB 1 via phosphorylation, which in turn activates mRNA translation [23]. Considering the amphipathic and positively charged nature of Orb2A's N-terminus, its involvement in LTM, and the high concentration of CaM in the neuron, we decided to investigate whether CaM might directly bind to Orb2A's N-terminus. Here we show that the N-terminal amphipathic domain of Orb2A indeed binds activated CaM and that this binding affects aggregation of Orb2A 1-88 into cross-β fibrils.

Protein expression and purification
All Orb2A 1-88 constructs, cloned into pET28b vectors, were described previously [8]. Wildtype Orb2A 1-88 ( Fig 1B) and cysteine mutants V6C, L18C, and G84C were expressed in E. coli Rosetta2 (DE3) cells (EMD Millipore), the other Orb2A 1-88 mutants were expressed using BL21 (DE3) cells. For all protein constructs, the appropriate plasmid was transformed into CaCl 2 chemically competent cells, and one colony was used to inoculate 25 ml of lysogeny broth (LB) Miller medium with the appropriate antibiotics at 37˚C. After approximately 4 h, this culture was diluted into 1 l LB with the appropriate antibiotics and was grown until OD 600 = 0.6. The culture was then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), and protein was expressed at 25˚C for approximately 16 h. Cell cultures were spun down at 4000 rpm in a Sorvall SLC-6000 rotor (Thermo Fisher Scientific, Waltham, MA) for 20 min at 4˚C, and the pellets were stored at -80˚C.

EPR studies
Protein aliquots were thawed on ice, and 2.5 μl of 40 mg/ml S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyr-rol-3-yl) methyl methanesulfonothioate (MTSL) spin label (Toronto Research Chemicals, North York, Ontario, Canada) was added (i.e. in great excess of the protein). The protein-MTSL mixture was incubated at room temperature for 1 h. Then the protein was diluted 15x with deionized water (dH 2 O) and added to a cation exchange column (S Ceramic HyperD F, Pall Life Sciences, Port Washington, NY), which had been equilibrated with dH 2 O. The flow-through was collected and the column was washed with 7 ml dH 2 O. The protein was then eluted with 2 ml of 8 M guanidine-HCl. This was then dialyzed 3x against 1l buffer with 20 mM HEPES, 100 mM NaCl, with or without 10 mM CaCl 2 , depending on if the experiment required Ca 2+ free conditions or not. Protein aggregates were centrifuged out of solution and the supernatant concentration was measured via its absorption at 280 nm. Finally, the desired amount of CaM, Ca 2+ , EDTA or buffer was added to the protein depending on the experiment, and the sample was loaded into a borosilicate capillary tube (0.6 mm inner diameter, 0.84 mm outer diameter; Vitro-Com, Mt. Lakes, NJ).
Continuous-wave EPR spectra were collected using a Bruker X-band EMX spectrometer (Bruker Biospin, Billerica, MA) at room temperature. For each spectrum 15 scans were accumulated in a high sensitivity cavity at a scan width of 150 gauss. The microwave power was 12.6 mW. Amplitude was calculated as the distance between the highest and lowest points of the spectrum. To determine the dissociation constant, amplitudes at different CaM concentrations were measured in triplicate and fitted to a one-site binding hyperbolic function using the method of least squares using an in house python script based on SciPy and matplotlib [24,25]. For kinetics curves, readings were taken of the specified sample over the indicated period of time. Amplitudes were normalized to the initial amplitude of each data set, and then plotted together for comparison.

Thioflavin-T fluorescence assays
Orb2A 1-88 protein aliquots were thawed on ice and the buffer was changed to 20 mM HEPES, 100 mM NaCl, and, depending on the experiment, 10 mM CaCl 2 using a PD10 desalting column. In a 96-well plate, Orb2A 1-88 was mixed with either buffer or CaM at a 1:1 molar ratio to a final concentration of 10 μM. Buffer and CaM by itself were run as controls. ThT was added to each well for a final concentration of 50 μM. Fluorescence kinetics were then acquired at room temperature with gentle periodic mixing in an Eppendorf (Hamburg, Germany) AF2200 plate reader using an excitation wavelength of 440 nm and emission wavelength of 480 nm. Error bars represent the standard deviation of three biological replicates.

Electron microscopy
Orb2A 1-88 protein aliquots were thawed, exchanged into 20 mM HEPES, 100 mM NaCl, and the appropriate amounts of Ca 2+ , EDTA, and CaM were added. In addition, 0.02% sodium azide was added to prevent microbial growth. Samples were incubated at room temperature with slight agitation. To image, copper formvar grids (Electron Microscopy Sciences, Hatfield, PA) were incubated with drops of protein sample for 5 min, and then incubated with uranyl acetate (1%) for another 5 min. Grids were then washed with two drops of uranyl acetate and one drop of water and allowed to dry. Finally, grids were imaged using a JEOL JEM-1400 EM (Tokyo, Japan).

Prediction of CaM binding sites
We initially applied various bioinformatics tools to see if they predicted the N-terminus of Orb2A to be a CaM binding domain in accordance with our hypothesis. Where the calmodulin target database [26] detected no CaM binding motif, the CaMELS algorithm [27] gave Orb2A 1-88 an overall interaction score of 0.68, but located the binding site at residue 65 rather than the N-terminus. In contrast, the calmodulin meta-analysis predictor [28] identified a potential CaM binding site at Orb2A residues 2-26, which showed similarities to the canonical 1-10, 1-5-10, 1-12, 1-14, and 1-16 binding motifs (see Fig 1D).

Orb2A 1-88 binds to activated CaM
Next, we wanted to test whether Orb2A indeed binds CaM. To do this, we measured EPR spectra of Orb2A 1-88 10R1 i.e. spin labeled at its natural cysteine. Residue 10 is located in the amphipathic sequence at the N-terminus of Orb2A which we hypothesized binds to CaM (Fig  1). In addition, using this wild-type cysteine as a reporter leads to minimal perturbation of the protein sequence.
After MTSL labeling, we acquired EPR spectra of Orb2A 1-88 10R1 in varying conditions: 1) Orb2A 1-88 10R1 alone; 2) Orb2A 1-88 10R1 in the presence of CaM and EDTA, which assures that CaM is in the inactive conformation; 3) Orb2A 1-88 10R1 in the presence of CaM and Ca 2+ , which assures that CaM is in its active conformation; 4) Orb2A 1-88 10R1 in the presence of Ca 2 + as a control. EPR line broadening is an indicator of rigidity at the site of the spin label and frequently used to measure protein binding [29]. As can be seen from Fig 2A, only in the presence of both CaM and Ca 2+ did we observe line broadening for Orb2A 1-88 10R1. Neither CaM in the presence of EDTA nor Ca 2+ alone caused line broadening suggesting that activated CaM binds the N-terminus of Orb2A.
Line broadening leads to a decrease in amplitude. We therefore used the EPR amplitude to estimate the fraction of Orb2A 1-88 binding to CaM in a concentration dependent binding curve. This is possible because, unlike linewidth, the EPR amplitude changes linearly with the fraction of bound protein if the unbound and fully bound state have different amplitudes (see supplement of [11]). We again labeled the wild type cysteine 10 with MTSL, and measured EPR spectra at different Orb2A 1-88 to CaM ratios (Fig 2B). From these data, we were also able to determine a dissociation constant of 1.6±0.5 μM.

Orb2A 1-88 binds to CaM within its N-terminal amphipathic domain
While the natural cysteine used for spin labeling in the above experiments was within the Nterminal amphipathic domain, we wanted to get a more specific idea of where on Orb2A 1-88 CaM was binding. We used cysteine mutants throughout Orb2A 1-88 for MTSL labeling which we described in previous publications [8,11]. We acquired EPR spectra in the presence of 10 mM Ca 2+ both with and without CaM and in a 1:1 molar ratio for each construct. Fig 3A   Fig 2. Activated CaM binds Orb2A 1-88 . A) EPR spectra of Orb2A 1-88 labeled at position 10 (10R1) show no change in linewidth with the addition of CaM together with EDTA or Ca 2+ alone. However, the linewidth increased with the addition of CaM together with Ca 2+ . CaM was added at a 2:1 molar ratio to Orb2A 1-88 , Ca 2+ and EDTA were added to a final concentration of 1 mM. EPR spectra intensity was normalized to their central linewidth. B) Change in EPR central line amplitude of 6 μM Orb2A 1-88 10R1 with increasing concentrations of CaM. Ca 2+ was present in excess (10 mM). Three biological replicates are shown together with a fit to a one-site binding hyperbolic function to determine the dissociation constant.

CaM binding inhibits aggregation of Orb2A 1-88
Previously, we showed that the cross-β core of fibrils formed by Orb2A 1-88 is located within the N-terminal amphipathic domain [8]. Considering that CaM binds to the same domain, we wanted to know whether CaM binding influenced Orb2A 1-88 fibril formation. We first used EPR to monitor amplitude changes over time in the N-terminal domain at 10R1 (i.e. the natural cysteine). Cross-β fibril formation usually results in a decrease in EPR amplitude [30]. Even though CaM binding already lowers the amplitude, we expect the amplitude to continue to decrease because fibril formation leads to very broad EPR lines at 10R1 due to electron spinexchange [8]. As can be seen from Fig 4, in the absence of CaM, we observed a decrease in EPR amplitude over two days as expected. However, in the presence of activated CaM, the EPR amplitude did not decrease over time but stayed relatively constant throughout the measurements. This observation indicates that Orb2A 1-88 bound to CaM is not forming an Nterminal cross-β fibril core.
We then wondered if Orb2A 1-88 was still forming fibrils but using a different domain as its cross-β core. For example via its Q/H-rich domain that was the core of ex vivo Orb2 fibrils or of the recombinant Orb2ΔRBD fragments that forms fibrils via phase separation [5,6]. To determine whether or not Orb2A 1-88 was aggregating in the Q/H-rich domain, we again used EPR to track the change in amplitude over time, but this time the MTLS label was at residue 34 (34R1) i.e. inside the Q/H-rich domain (Fig 4). We observed a slight decrease in amplitude over time for Orb2A 1-88 without CaM, but we saw no decrease in amplitude in the presence of CaM. This confirms our initial conclusion that without CaM, Orb2A 1-88 forms fibrils with a cross-β core at its N-terminus, but in the presence of CaM, Orb2A 1-88 does not aggregate.

The aggregation inhibited by CaM is cross-β in nature
To confirm that CaM inhibits fibril formation of Orb2A 1-88 , we used Thioflavin-T (ThT) fluorescence, which is commonly used as an indicator of cross-β fibril formation [31]. It is thought that ThT binds to the β-sheet core of a fibril, which stabilizes the rotating bond and causes fluorescence [32,33]. Because of this, the change in ThT fluorescence can be used to measure fibril forming kinetics. We tracked the ThT fluorescence over time for both Orb2A 1-88 alone, and Orb2A 1-88 with CaM ( Fig 5A). Orb2A 1-88 alone gave a characteristic cross-β aggregation curve with a lag phase followed by a sigmoidal increase in fluorescence, and a plateau after about 4-5 days. This increase in Tht fluorescence is somewhat slower than the decrease of EPR amplitude in our 10R1 sample. For Orb2A 1-88 samples where CaM and Ca 2+ were present, we did not observe any net change in ThT fluorescence over time. However, the ThT fluorescence of samples with activated CaM was quite high even in the absence of Orb2A 1-88 . We speculate that this is caused by activated CaM binding ThT in its hydrophobic binding pockets.
To confirm these ThT fluorescence results, we used electron microscopy (EM). We saw that while the ThT positive Orb2A 1-88 sample contained visible fibrils, Orb2A 1-88 with CaM and Ca 2+ did not form any structures resembling fibrils (Fig 5B). This supports the conclusion that CaM inhibits cross-β aggregation of Orb2A 1-88 .

Discussion
In this study, we show that the first 88 amino acids of Orb2A bind to CaM via their N-terminal domain. We previously showed that this domain is able to form an amphipathic helix that is capable of binding anionic lipid membranes, and proposed that Orb2A 1-88 preferred anionic lipid vesicles because of the lysines present at position 4 and 20, as well as the positively charged N-terminus [11]. Positively charged amphipathic helices have also been shown to be preferred binding partners for CaM [34]. In addition, one CaM binding site prediction algorithm identified the N-terminus of Orb2A as a potential binding site. Our EPR data confirmed this hypothesis showing that calcium activated CaM can bind to the N-terminus of Orb2A 1-88 . In addition, we showed that this interaction prevented the formation of Orb2A 1-88 fibrils.
Our EPR, ThT fluorimetry and EM data show that CaM inhibits Orb2A 1-88 fibril formation in the presence of Ca 2+ . This was expected because the N-terminal amphipathic domain that interacts with CaM also forms the cross-β core of Orb2A 1-88 fibrils [8]. Previously, we showed that the binding of the N-terminal amphipathic domain to lipid membranes also inhibited fibril formation [11]. We hypothesized that binding to lipid membranes stabilized an N-terminal helix thereby not allowing the same domain to form a cross-β structure. CaM binding of Orb2A 1-88 could create a similar situation where the N-terminus is stabilized in a helical conformation unable to interact with other Orb2A 1-88 monomers to form a cross-β fibril (see Fig 6).
Although our data show that the CaM-Orb2A 1-88 interaction is specific and long-lasting, the dissociation constant we determined (1.6 μM) is higher than for most CaM targets which have Kds in the range of 10 −7 to 10 −11 M [35]. However, there are several reports of similar or even higher dissociation constants in the literature [36,37].
Calcium signaling is very important for LTM, and CaM is an integral mediator of Ca 2+ signaling inside neurons. A key function of CaM in this context is to bind and activate the kinase CaMKII and other enzymes that are important for synaptic plasticity [12,[14][15][16][17][18][19][20]. Interestingly, some CPEB homologues, such as CPEB-1 in mice and CPEB in Xenopus oocytes, are activated when phosphorylated by CaMKII [23,38]. Although White-Grindley and co-workers showed that phosphorylation of Orb2A induced long-term memory by increasing its half life, it is the Tob (Transducer of Erb-B2) dependent phosphorylation of Orb2 via the Lim Kinase rather than CaMKII that is responsible for this effect [39]. It is interesting to speculate that in the case of Orb2A, activation could happen by the direct interaction with CaM, thereby circumventing the extra step of phosphorylation via CaMKII.
However, we do not know if and how binding of CaM to the N-terminus specific to Orb2A can affect learning and memory in vivo. In addition, why would CaM binding to Orb2A 1-88 prevent its fibril formation if both Ca 2+ activated CaM and Orb2 aggregation are necessary for long-term memory? The answer to this question might come from the fact that although the N-terminal, amphipathic domain of Orb2 is important for initiating Orb2 fibril formation necessary for LTM [2,3,7], the fibril core of functional Orb2 fibrils is located in the Q/H-rich domain that is shared between Orb2A and B rather than the N-terminus specific to Orb2A [5]. Our previous work has shown that the N-terminus of Orb2A can form cross-β fibrils on its own in Orb2A 1-88 . In this construct, Q/H-rich fibril core of Orb2 was not observed. On the other hand, Orb2AΔRBD fibrils that were made via phase separation showed the presence of the Q/H-rich fibril core rather than the N-terminal core [6], suggesting that these cores might be mutually exclusive. Similarly, mutually exclusive fibril cores were recently reported for TDP-43 [40]. In a scenario where only either the N-terminal or the Q/H-rich core of Orb2A can exist, the binding of CaM could facilitate the formation of the latter by preventing the formation of the former.
Another solution to the apparent contradiction that CaM interaction prevents fibril formation could have something to do with the fact that Orb2 aggregation is only associated with LTP maintenance and not initiation. For example, CaMKII activation becomes independent from CaM binding when the cell switches from LTP initiation to LTP maintenance. Similarly, Orb2A aggregation could initially be inhibited by CaM binding but move forward at a later point to form Orb2 fibrils important for LTP maintenance but not initiation.
Both hypotheses are compatible with our previous proposal that the cross-β core found within the N-terminal amphipathic domain of Orb2A is potentially transient or regulatory for the more permanent Q/H-rich core found in functional Orb2 in vivo [5,8]. More studies are needed to fully understand the molecular processes involved in LTM in general, and particularly on the regulation of Orb2A fibrillization in cells and in vivo. Supporting information S1 Data. Data from graphs found in the paper. Plot_Data_Orb2-CaM_interaction. (XLSX)